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Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Goldfless SJ, Belmont BJ, de Paz AM, Liu JF, Niles JC - Nucleic Acids Res. (2012)

Bottom Line: Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation.By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

ABSTRACT
Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

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Population measurements of yeast gene expression regulated by TetR aptamers.Flow cytometry histograms show population-wide expression levels of aptamer-regulated vYFP. The aptamer located within the 5′-UTR of vYFP and the expression status of TetR are indicated. In (a), the vYFP reporter is episomal, and in (b) integrated at the TRP1 locus. Shaded gray histograms represent the auto-fluorescence of the background yeast strain. Measurements for yeast grown in the absence (red) and presence (blue) of Dox are indicated. Data for each histogram are representative of four independent experiments.
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gks028-F3: Population measurements of yeast gene expression regulated by TetR aptamers.Flow cytometry histograms show population-wide expression levels of aptamer-regulated vYFP. The aptamer located within the 5′-UTR of vYFP and the expression status of TetR are indicated. In (a), the vYFP reporter is episomal, and in (b) integrated at the TRP1 locus. Shaded gray histograms represent the auto-fluorescence of the background yeast strain. Measurements for yeast grown in the absence (red) and presence (blue) of Dox are indicated. Data for each histogram are representative of four independent experiments.

Mentions: We performed RRL translation regulation experiments using fixed mRNA and titrated TetR concentrations. Translation of mRNA containing aptamer 5–1.2, but not 5–1.2m2, was dose-dependently repressed by TetR, reaching ∼70% repression at a 5:1 TetR:mRNA ratio (Figure 2c). This indicates that TetR-dependent translational repression is specific to mRNA containing an aptamer competent for binding TetR and definitively occurs post-transcriptionally. Repression was fully relieved by Dox (22 μM), consistent with inducibility of the TetR–aptamer interaction. Next, we tested the specificity of inducible translation in yeast using the experimental design described above, but including either a non-functional RNA (5–1.2m2) or an unrelated protein (the iron-responsive element binding protein, IRP) as substitutes for 5–1.2 and TetR, respectively (Figure 2d). We observed aTc-inducible regulation of FLuc synthesis (∼80%) only in strains simultaneously carrying 5–1.2 and expressing TetR. Importantly, TetR was only expressed in galactose-containing media and its abundance was not decreased by aTc, as confirmed by western blot (Supplementary Figure S1). For the above experiments, we used galactose-inducible transcription to control TetR expression, allowing us to test isogenic strains. However, even when TetR was expressed constitutively from the TDH3 promoter, we found identical Tc-dependent translational regulation (Supplementary Figure S2). These data underscore the potential for using this system in biological contexts where transcriptional control is not accessible. qPCR analysis showed that increased FLuc reporter expression in the presence of aTc was not accompanied by an increase in FLuc mRNA, as would be expected if a transcriptional response or a significant change in mRNA stability were responsible for inducible expression (Supplementary Figure S3). Altogether, these data demonstrate that the observed in vivo regulation occurs at the translational level and is due to a specific interaction between TetR and an RNA aptamer competent for binding TetR. Lastly, we replaced FLuc with Venus yellow fluorescent protein (vYFP), and this reporter was either expressed episomally or integrated at the TRP1 locus. Flow cytometry showed quantitatively that inducible expression is homogeneous across a yeast cell population, and similar in dynamic range irrespective of the gene being regulated (Figure 3).Figure 3.


Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Goldfless SJ, Belmont BJ, de Paz AM, Liu JF, Niles JC - Nucleic Acids Res. (2012)

Population measurements of yeast gene expression regulated by TetR aptamers.Flow cytometry histograms show population-wide expression levels of aptamer-regulated vYFP. The aptamer located within the 5′-UTR of vYFP and the expression status of TetR are indicated. In (a), the vYFP reporter is episomal, and in (b) integrated at the TRP1 locus. Shaded gray histograms represent the auto-fluorescence of the background yeast strain. Measurements for yeast grown in the absence (red) and presence (blue) of Dox are indicated. Data for each histogram are representative of four independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351163&req=5

gks028-F3: Population measurements of yeast gene expression regulated by TetR aptamers.Flow cytometry histograms show population-wide expression levels of aptamer-regulated vYFP. The aptamer located within the 5′-UTR of vYFP and the expression status of TetR are indicated. In (a), the vYFP reporter is episomal, and in (b) integrated at the TRP1 locus. Shaded gray histograms represent the auto-fluorescence of the background yeast strain. Measurements for yeast grown in the absence (red) and presence (blue) of Dox are indicated. Data for each histogram are representative of four independent experiments.
Mentions: We performed RRL translation regulation experiments using fixed mRNA and titrated TetR concentrations. Translation of mRNA containing aptamer 5–1.2, but not 5–1.2m2, was dose-dependently repressed by TetR, reaching ∼70% repression at a 5:1 TetR:mRNA ratio (Figure 2c). This indicates that TetR-dependent translational repression is specific to mRNA containing an aptamer competent for binding TetR and definitively occurs post-transcriptionally. Repression was fully relieved by Dox (22 μM), consistent with inducibility of the TetR–aptamer interaction. Next, we tested the specificity of inducible translation in yeast using the experimental design described above, but including either a non-functional RNA (5–1.2m2) or an unrelated protein (the iron-responsive element binding protein, IRP) as substitutes for 5–1.2 and TetR, respectively (Figure 2d). We observed aTc-inducible regulation of FLuc synthesis (∼80%) only in strains simultaneously carrying 5–1.2 and expressing TetR. Importantly, TetR was only expressed in galactose-containing media and its abundance was not decreased by aTc, as confirmed by western blot (Supplementary Figure S1). For the above experiments, we used galactose-inducible transcription to control TetR expression, allowing us to test isogenic strains. However, even when TetR was expressed constitutively from the TDH3 promoter, we found identical Tc-dependent translational regulation (Supplementary Figure S2). These data underscore the potential for using this system in biological contexts where transcriptional control is not accessible. qPCR analysis showed that increased FLuc reporter expression in the presence of aTc was not accompanied by an increase in FLuc mRNA, as would be expected if a transcriptional response or a significant change in mRNA stability were responsible for inducible expression (Supplementary Figure S3). Altogether, these data demonstrate that the observed in vivo regulation occurs at the translational level and is due to a specific interaction between TetR and an RNA aptamer competent for binding TetR. Lastly, we replaced FLuc with Venus yellow fluorescent protein (vYFP), and this reporter was either expressed episomally or integrated at the TRP1 locus. Flow cytometry showed quantitatively that inducible expression is homogeneous across a yeast cell population, and similar in dynamic range irrespective of the gene being regulated (Figure 3).Figure 3.

Bottom Line: Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation.By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

ABSTRACT
Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

Show MeSH
Related in: MedlinePlus